The conjugation of ubiquitin to a protein target, or ubiquitination, is an essential component across a wide array of cellular process including degradation, DNA repair, endocytosis, and protein localization. It is accomplished through a tightly regulated series of enzymatic steps;with the most important being the direct transfer of ubiquitin to an internal lysine residue by E3 ubiquitin ligases. Target-ligase binding is mediated by the recognition of specific amino acid degradation sequences, or degrons. Improper regulation of either the E3 ligases or their target proteins has been implicated in the development of cancers such as acute myeloid leukemia (AML), in fact, targeting of the ubiquitin-proteasome pathway is one of the leading directions of AML chemotherapeutics. The work proposed herein deals with the design, synthesis and evaluation of degron-based substrates as a novel E3 ligase reporting mechanism. Aim 1 will utilize standard FMOC synthesis techniques to construct a small catalog (approximately twenty-five) of substrates, each consisting of four essential components 1) the degron, 2) a proximal lysine residue to be ubiquitinated, 3) a linker to avoid steric hindrance issues, and 4) a fluorescein tag to serve as a visual marker. In Aim 2, substrate ubiquitination will be evaluated using S100 cytosolic lysates first in a model cell system and secondly in myeloid leukemia-based cells such U937, THP-1, K562, NB4, Kcl-22, and KU812 using both gel and capillary electrophoresis. These assays will be utilized to generate conclusions regarding simple kinetic parameters based on Michaelis-Menten kinetics as well as the relative substrate half life, ultimately developing a metric to rank substrate ubiquitination. Aim 3 will utilize this metric to select a small number (less than five) of substrates to be tested as E3 ligase reporters in a single-cell system. A rigorous study will be conducted to determine the ideal method, ranging from electroporation to myristoylation, to insert the substrate into one of the aforementioned cell lines. Finally, the reporters will be studied in a single-cell capillary electrophoresis system capable of lysing and detecting substrate ubiquitination. Ultimately, this proposal aims to gain a greater understanding of protein ubiquitination by E3 ubiquitin ligases through the creation of a new reporting mechanism that is compatible with a single-cell system and capable of evaluating intracellular ubiquitination. PUBLIC HEALTH RELEVANCE: The proposed research project will focus on the development of peptides representative of essential proteins that are improperly regulated during acute myeloid leukemia (AML). Further, these peptide substrates will be used as a novel reporting mechanism to evaluate protein ubiquitination in AML-related immortal cell lines using a single-cell capillary electrophoresis tool.